The invention relates generally to the synthesis and use of colloidal organosilica particles, and, more particularly, to compositions and methods for synthesis of controlled size silica that allow binding of activated molecules for diagnostics and other uses.
Rapid genetic sequencing holds the key to understanding the fundamental processes which govern cellular and molecular biology. Gene discovery has been the driving force of biotechnology over the past decade, but to obtain a better understanding of how genes are regulated and expressed in different individuals, the genetic sequence is simply not enough. It is necessary to be able to identify changes in single base pairs (Single Nucleotide Polymorphism—SNP) within a gene to understand why and how genes are expressed differently in different individuals. DNA sequencing of this magnitude can only be contemplated when sequencing techniques have been made considerably more cost-effective and robust than they are today, or will be in the foreseeable future.
The ability to perform DNA sequencing with an extremely large number of probes (e.g., 1010) on an inexpensive and well-defined high throughput platform is highly desirable and would overcome major deficiencies in current technology. The existing techniques used for sequencing genetic information (e.g., DNA microarray devices) are limited by speed, cost, and library size. Colloid-based libraries, however, are emerging as an extremely attractive alternative because they use significantly smaller probe sites (colloidal particles rather than spatially resolved sites on a microarray) and a 3-dimensional configuration. Instead of preparing oligonucleotide probes in pixels on a microarray, the probes can be either attached to (or synthesized on) polymer or ceramic particles 2 to 300 μm in diameter. Some of the most impressive advantages of colloids are that they are inexpensive to produce in enormous numbers and they can be conveniently stored in small volumes of fluid. Another important advantage, which has not been fully utilized to date, is the ability to generate enormous random libraries of probes via combinatorial split-and-mix processes (FIG. 1). The colloids required for this purpose should be encoded to permit each probe to be distinguished. The strategy requires colloidal supports which have unique and reproducible optical signatures (i.e., fluorescence and light scattering attributes) as detected, e.g., by a high performance flow cytometer.
Microparticles
Microparticles, having diameters measured in microns, are important in the biotechnology industry, and have assumed a special role as supports for synthesis of polymers such as oligonucleotides. Useful particles have been formed from organic polymers, such as polystyrene cross-linked with divinylbenzene, and have found use in combinatorial chemistry, which will play a growing role in developing new drugs and diagnostics. Such particles often swell in solvents. This swelling is another variable to consider when using the particles in solvents during reactions such as coupling reactions that occur on particles used in nucleic acid synthesis.
Typically, in combinatorial synthesis, particle swelling desirably increases particle surface area. However, when used for colloidal sequencing reactions the particle's optical properties are important for indicating the sequence of the polymer, such as an oligonucleotide, coupled to the surface. Particle swelling thus alters the optical properties and may jeopardize the encoded information. Another problem, particularly seen with commercially available polystyrene particles, is that dyes incorporated into the particles leach out when the particles are exposed to certain solvents.
Silica spherical particles are an improvement to this technology in some aspects. Silicon based particles resist swelling in solvents and generally display more consistent optical properties during their use in chemical procedures such as polymer synthesis. Unfortunately, commercially available silica particles typically are limited to less than 5 microns diameter size. Such sizes are too small for many flow cytometer applications and for use in machinery such as a conventional DNA synthesizer.
The Stober Process
A common method for synthesizing colloidal silica is the Stober process, which is a specific application of sol-gel synthesis as described by Stober et al. J. Colloid Interface Sci. 26, 62 (1968) and diagrammed in FIG. 2. The first step in the Stober process as shown in this figure is the hydrolysis of tetraethyl orthosilicate (TEOS) in a solution of ethanol, water, and ammonia as described by Brinker et al., J. Non-Cryst. Solids 48, 47-64 (1982). In this step, ammonia catalyzes the hydrolysis of TEOS (I) to form reactive silanol groups and hydroxyl (II). In a second base catalyzed reaction, the silanol groups (III, IV) condense to form a polymer chain (V).
As the polymer chain increases in length from the two reaction steps, the polymer solubility decreases until the chain no longer dissolves in the solution as described by Bogush et al. J. Colloid Interface Sci. 142, 1-18 (1991). The polymer precipitates out of solution as nano-sized silica particles, which are colloidally unstable. Consequently, the particles aggregate to form larger particles, as described by Bogush et al., J. Colloid Interface Sci. 142, 19-34 (1991). The particles formed from this process are monodisperse because the aggregation rate between a large aggregated particle and a nano-sized particle is much greater than the aggregation rate between two large aggregates or two nano-sized particles. This relationship is explained by Bogush et al., J. Non-Cryst. Solids 104, 95-106 (1988).
The Stober process outlined above advantageously allows incorporation of fluorescent dyes into the silica network. This incorporation may occur via use of silane coupling agents, such as 3-aminopropyl trimethoxysilane (APS), which react with isothiocyanate modified dyes to form fluorescent silane monomers as shown in FIG. 3. See for example van Blaaderen et al. Langmuir 8, 2921-2931 (1992). The methoxy groups hydrolyze to form silanol groups. The silanols subsequently condense with TEOS (see FIG. 2) and are incorporated into the polymer chains.
Unfortunately, particles formed via the Stober process generally are limited to a maximum size of approximately 3 microns, which is insufficient for many purposes. Particle sizes desirably should be controlled over a larger diameter range. Further, the particles generally have limited porosities yet many procedures require use of or could benefit from large porosity particles. Such an improvement in particles and methods for their synthesis would allow greater commercial use of desirable silicon-based particles.
Acid Catalyzed Processes
Acid catalyzed hydrolysis of TEOS provides an alternative mechanism for forming colloidal silica as described by many workers (Kawaguchi and Ono J. Non-Cryst. Solids 121, 383-388 (1990); Karmakar et al. J. Non-Cryst. Solids 135, 29-36 (1991); Ding and Day J. Mater. Res. 6, 168-174 (1991); Mon et al. J. Cer. Soc. Jap. 101,1149-1151(1993); Ono and Takahashi World Congress on Particle Technology 3, 20 1-11; Pope Mater. Res. Soc. Symp. Proc. 372, 253-262 (1995) and Pope, SPIE, 1758, 360-371 (1992). Many methods have been employed using acid hydrolysis to form silica films (Aksay et al., Science 273, 892-898 (1996)) and non-spherical particles (Soten and Ozin Curr. Opin. Colloid Interface Sci. 4, 325-337 (1999)), but the method of acid synthesis formation of spherical particles is less well developed.
More recent reports of acid catalyzed particle formation show the formation of uniform pure silica spheres of up to approximately 10 microns. See Yang et al., Journal of Materials Chemistry 8, 743-750 (1998); Qi et al., Chem. Mater. 10, 1623-1626 (1998); and Boissiere and Lee Chemical Communications 2047-2048 (1999). However, these spherical particles generally require covalent linking of dye into the silica network for practical applications. For this reason silane coupling agents such as APS and 3-mercaptopropyl trimethoxysilane (MPS) often are chosen for incorporation into the particles. Yet another problem generally with these particles is the limitation of the number of binding sites on their surfaces and limited porosity, which often dominate the usefulness of the particles. Thus, any method that can increase the number of binding sites and/or porosity would be beneficial in a number of fields of use.
One particularly important field of use that would benefit greatly from use of particles having a high binding site density is colloidal sequencing. Colloidal sequencing uses particles as supports to build molecules formed by diverse sequential reactions, and requires a method (typically optical) for labeling the different species formed. Unfortunately, such particles are not easily optically distinguishable from each other. Most commercially available particles contain only up to 3 dyes, and the dyes are incorporated in equal amounts. Applications such as colloidal sequencing generally require the use of multiple dyes in different ratios for tagging different particle types and to create optical diversity. Furthermore, the particles used for colloidal sequencing must be compatible with organic solvents and with reaction conditions used for polymer formation. Accordingly, large stable particles having a high number of binding sides and/or porosity would be very useful for this art.